16 well e plate

Similarly to the migration assays, the effect of the SP2024 peptide on cellular adhesion was assessed using the RT-CIM technology. In this case, 25,000 cells/well were plated in 16-well E-plates (Roche, Basel, Switzerland) in the presence or absence of the peptide. The adhesion was monitored over/for 3 hours) by measuring changes in the electrical impedance, which is a direct measure of the cells adhering on/to the electrodes. All in vitro studies used as control full supplemented media with equivalent DMSO concentration as in the treatment samples but maintained to levels less than 0.2%.

U87 MG (ATCC HTB-14^TM) glioma cells were cultured at 37°C in 5% CO₂ atmospheric pressure in DMEM supplemented with 10% FCS. Cells were plated in 16-well e-plates (Roche, Hungary), 96-well culture plates, T25 or T100 flasks at various densities depending on the type and set-up of the experiment. UFAs were administered 1 hour prior to irradiation. The added UFAs were present in the medium throughout the whole incubation interval, 24, 48, 72 or 100 hours, respectively. The UFAs could incorporate into the membranes or enter the cell until the end of the experiment. Cells were treated with the following UFAs: AA (Cayman Chemical Company, San Diego, California), EPA (Sigma-Aldrich, Budapest, Hungary), DHA (Cayman Chemical Company), GLA (Ubichem Research, Budapest, Hungary), OA (Sigma-Aldrich). Then cells were subjected to a dose of 5 or 10 Gy and incubated for 24, 48 or 72 hours.

Cell proliferation was determined using the xCELLigence RTCA system (Roche, Basle, Suizerland). Real-time recording of cell proliferation was started 24 h after transfection with the indicated siRNAs. In EGFR inhibition assays, cells were seeded in a medium containing the indicated concentration of Erlotinib HCI (OSI-774 HCI, Selleck Chemicals GmbH (Cologne, Germany), #S1023). Cells were seeded in 16-well E-plates (Roche) at the following densities: VM-CUB-1 (2500 cells/well); 5637, CAL-29, HT-1376, UM-UC-6, UM-UC-10, T24, TCC-SUP-G, J82, and RT-112 cells (all at 5000 cells/well); UM-UC-3 (7500 cells/well); and JON (10,000 cells/well). The system recorded cell indices at 15 min intervals.

To exclude a distinct influence of fluoresceinamine-labeled (F-) heparin on cell proliferation, BM-, UC- and WAT-STCs were cultured in the presence of unlabeled and F-heparin. Experiments were performed by xCELLigence RTCA DP instrument (Roche Diagnostics) placed in a humidified incubator at 37 °C and ambient air conditions. After an incubation of cell-free growth medium for 30 minutes at room temperature, the background impedance was determined. For each measurement, 1 × 10³ cells per well were seeded in the presence of either 2 IU/mL standard heparin (Biochrom) or F-heparin (PG Research) into 16-well E-plates (Roche Diagnostics) in quadruplicates. To allow cell attachment, E-plates were stored at room temperature for 30 minutes. The E-plates were then locked in the RTCA DP device, the impedance value of each well was automatically monitored by the xCELLigence system and expressed as cell index (CI) value. Cells were cultured for 96–98 hours with CI monitoring every 5 minutes.

The xCELLigence
RTCA system (Roche) was employed to assess the impact of blocking
A_2BAR-mediated effects in cellular impedance upon receptor
activation, as previously described.^{57 (link)−60 (link)} To this end, A_2BAR^SNAP cells were growth in 16-well E-plates (Roche), using DMEM
supplemented with 1 mM sodium pyruvate, 2 mM l-glutamine,
100 U/mL streptomycin, 100 mg/mL penicillin, and 1.5% (v/v) fetal
bovine serum in the presence of 0.5 U/mL of ADA. Of note, wells were
previously coated with 50 μL fibronectin (10 μg/mL; Sigma-Aldrich)
and the background index for each well was determined with supplemented
DMEM (90 μL) in the absence of cells. Subsequently, A_2BAR^SNAP cells (90 μL) were plated at a cell density
of 10,000 cells/well and grown for 18 h in the RTCA SP device station
(Roche) at 37 °C in an atmosphere of 5% CO₂. Then,
before ligand addition, cell index values were normalized to the same
time point using the RTCA software, providing the normalized cell
index (NCI). After ligand stimulation, NCI was recorded every 15 s
for a total time of at least 45 min. The area under the curve (AUC)
for every condition was calculated using GraphPad Prism 9.

The xCELLigence (RTCA) system was used to monitor cell behavior throughout the experiment. The system monitors changes in morphology, adherence and proliferation after exposition to tested substances, and has been described in many works [36 (link),37 (link)]. In our experiment, cells were inoculated into 16-well E-plates (Roche Applied Science, Mannheim, Germany) with density 10,000 cells/well. After initial 20-h cultivation, the cells were still within a log phase and formed an 80% monolayer. The tested substances were added to the cells at the final concentration of 0.1–100 µg/mL (E-2–E-5) calculated in triplicates and incubated for an additional 48 h. Cell changes were measured in real time each hour and recorded in a dimensionless unit cell index (CI). The more cells attached to the bottom of the well, the higher adherence, proliferation and CI activity. Proliferative activity (PA) was expressed as a percentage using control cells without treatment (100%) according to the following formula: % PA = CI_sample × 100/CI_control.